This project focuses on the human cystic fibrosis transmembrane conductance regulator (CFTR), with the overall goals of (l) providing a map of the conduction pathway and (2) determining the interaction between the R-domain and this pathway. The major experimental tool is electrophysiological measurements on wild-type and mutant human CFTR expressed in Xenopus oocytes. (l) The ion channel will be mapped as follows. Based upon the recent characterization of the voltage-dependent blocking action of two acidic inhibitors of the wild-type CFTR -- diphenylamine-2-carboxylate (DPC) and flufenamic acid (FFA) --site-directed mutant channels are being designed and constructed. The mutant channels will be tested with respect to blockade and single-channel conductance, with the goal of localizing the amino acid residues that line the pore. Preliminary studies have outlined interesting regions to investigate. (2) The project will also map the contacting surfaces between the regulatory (R) domain and the bulk of the channel using the same methods that have proven successful for Specific Aim (l). Site-directed mutations will be made within (a) portions of the R-domain that may serve as donor regions and (b) portions of the bulk of the channel that may serve as acceptor regions. These mutations will be studied with respect to kinetics of activation/deactivation, in order to understand the mechanism of gating within CFTR. In parallel experiments, data will be used from two additional sources: construction of chimeric channels from CFTR/P- glycoprotein sequences (since the P-glycoprotein does not include an R- domain) and from human/shark CFTR sequences (if gating kinetics in the shark channel differ substantially from those of the human). These studies will provide an increased understanding of the function of CFTR, and may lead to new therapeutic strategies.